Hydrogen exchange (HX) methods are being used to study allosteric process in hemoglobin. Difference HX methods, developed earlier, make it possible to label selectively with tritium just those exchangeable peptide NH sites on protein segments that are sensitive to the functional interaction being studied. The position of the label can then be determined by use of the fragmentation-separation method. The latter has been improved to allow quantitative results to be obtained. These methods together provide a "functional labeling" approach that may be able to measure the parts of any protein that are involved in its functional interactions and the contribution in terms of structural free energy that each involved segment makes to that function. Also with these methods one can study a definable aspect of the internal dynamic behavior of proteins. These methods will be further improved and used in an attempt to identify and study the properties of all the allosterically sensitive sites in hemoglobin and some of the allosterically insensitive sites. The aim is to map out the whole allosterically involved region of the hemoglobin molecule and to evaluate the structural energy relationships that shape the allosteric process. This work will also provide direct information concerning the kinds of internal protein motions that determine protein H-exchange behavior. The work proposed promises to add previously unavailable kinds of information to our understanding of the allosteric phenomenon in hemoglobin, which has immediate implications for the way in which the oxygen delivery process is controlled and for particular derangements in this process, as in hemoglobin mutations. More generally this basic molecular mechanism, though it occurs nearly universally in biological regulation, is not understood in any single case, and hemoglobin presents a favorable case for working out the principles of these systems.